To study deoxyribonucleoside triphosphate metabolism during the cell cycle, we conducted experiments to enrich cultures for specific cell cycle populations. With dibutyryl cyclic AMP and theophylline, S49 cells are reversibly arrested in G1 phase. Washout of dibutyryl cyclic AMP and theophylline gradually releases cells from the G1 block and, by 24 hours post-washout, approximately 60% of the cells are in S phase. Colcemid (10 micrograms/ml for 12 hours) yields cultures enriched with cells in G2/M (70%), as assessed by cytofluorimetry. G1 cells have relatively low pools of dCTP and dTTP and relatively high pools of dATP. These pool ratios are reversed in S phase, suggesting that allosteric inhibition of ribonucleotide reductase by high pools of dATP is operating to lower dCTP and dTTP pools in G1 phase. Ribonucleotide reductase activity when measured in the absence of allosteric effector molecules is increased in S phase suggesting that a mechanism other than allosteric activation is involved in the S phase, activation of ribonucleotide reductase. We investigated the possibility that increased synthesis of the M1 subunit of ribonucleotide reductase was responsible for the increased activity. The M1 subunit was identified by two-dimensional gel electrophoresis of [35S]methionine-labeled whole cell extracts by coelectrophoresing the extracts with purified protein. We were unable to detect a significant increase in the quantity of M1 during S phase. Experiments are in progress to determine whether or not the M2 subunit is in greater abundance during S phase.